| dc.description.abstract | Alginate is a polysaccharide found in nature as structural component in marine brown algae and as capsular polysaccharide in some soil bacteria. Alginates have the potential to work as immobilization material for cells, providing a mild encapsulation protocol by ionic cross-linking at physiological conditions. By encapsulating the cells into alginate gel spheres, the alginate gel can serve as protection against the host immune system upon transplantation of therapeutic cells. This may be useful in the treatment of different diseases, like diabetes mellitus and brain tumours.
For alginate to work as immobilization material for cells, high demands are required for the capsules stability, permeability and biocompatibility. The composition of the alginate, consisting of mannuronic acid and guluronic acid, are important for the alginate capsule properties. By modifying the structure by the use of mannuronan C-5 epimerases in vitro, it is possible to tailor the alginate to suit the desired function. Recent studies have shown that alginate capsules of epimerized alginate consisting of solely MG- and G-blocks, is less permeable to IgG and more stable than alginate capsules made of natural high-G alginate. These characteristics should make the epimerized alginate capsule more suitable as immobilization material for cell transplantation.
In this study mannuronan C-5 epimerases, AlgE4 and AlgE1, were used to epimerize a high-M alginate, converting it to an alginate consisting of solely MG- and G-blocks. The epimerized alginate was purified by the use of active coal filtration and NaOH treatment, and capsule properties with regard to size, permeability and inhomogeneity was investigated. Next, 293 endo cells and human islets of Langerhans were encapsulated in epimerized alginate capsules (EpiAlg) and natural high-G alginate capsules (TAM) for comparison of cellular growth and viability.
The capsule size was measured in a conventional light microscope and revealed that EpiAlg had a smaller size compared to TAM. A smaller size may be a benefit as the surface to volume ratio increases. The EpiAlg capsules also showed a slightly higher degree of inhomogeneity, but no difference was seen in the permeability between the two capsule types. A slightly slower diffusion of IgG was observed for the epimerized alginate with the lowest G content (64%) compared to the epimerized alginate with a higher G content (71%).
293 endo cells encapsulated in EpiAlg and TAM capsules were examined for metabolic activity by a MTT assay, viability by a live/dead assay and morphology by histology cross sections. Cells encapsulated in EpiAlg gelled with only Ca2+ showed a higher metabolism compared to cells encapsulated in TAM gelled with Ca2+/Ba2+. Under these conditions, however, EpiAlg (Ca2+) was less stable than TAM (Ca2+/Ba2+). Histology sections of 293 endo cells encapsulated in EpiAlg (Ca2+) and TAM (Ca2+/Ba2+), showed that the cells grow in denser clusters in TAM (Ca2+/Ba2+) compared to EpiAlg (Ca2+). A second encapsulation showed that 293 endo cells encapsulated in EpiAlg gelled with Ca2+/Ba2+ had a lower metabolism compared to cells encapsulated in TAM gelled with Ca2+/Ba2+. During this experiment TAM capsules seemed to rupture more easily than EpiAlg capsules. No differences were seen in the confocal images with regard to live and dead cells between the two capsule types.
Human islets of Langerhans were encapsulated in EpiAlg and TAM, to compare the islets viability in the two capsule types. A static incubation assay was performed to look at the response of the islets when stimulated with low glucose solution and high glucose solution. An ELISA kit was used to measure the insulin production. The results showed low stimulation indexes for both capsule types.
However, the low viability of the islets already from before encapsulation probably affected the results. Earlier studies have shown that islets encapsulated in TAM, are viable and produce insulin 140 days after encapsulation.
Human islets of Langerhans were also encapsulated in TAM and EpiAlg with fluoresceinamine labelled alginate. The islets showed an intense green fluorescence, which seemed to be due to auto fluorescence of the islets. | nb_NO |
